DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-2, 4-5, 7-9, 11-12, 14 and 16-25 are pending.
Claims 16-25 are withdrawn.
Claims 1-2, 4-5, 7-9, 11-12, and 14 are under examination.
Election/Restrictions
Applicant’s election without traverse of the following invention
Invention
Group I, claims 1-2, 4-5, 7-9, 11-12, and 14, drawn to a method of transfecting a human macrophage with genetic material, the method comprising the steps of: (a) contacting a human macrophage with genetic material; (b) electroporating the macrophage with a first pulse phase, wherein the first pulse phase comprises a burst of unipolar pulses or a square pulse, wherein each pulse is between 750- 1000V, and wherein the first pulse phase lasts for a total period of between 20- 500μs; and (c) electroporating the macrophage with a second pulse phase, wherein the second pulse
phase comprises a burst of pulses or a square pulse, wherein each pulse is between 50- 225V,
and wherein the second pulse phase lasts for a total period of 2000-50000 μs,
in the reply filed on 8th, October, 2025 is acknowledged.
The requirement is still deemed proper and is therefore made FINAL.
Claims 16-25 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claim Objections
Claims 8 and 12 are objected to because of the following informalities:
Claim 8 does not end with a period. Applicant is directed to MPEP 608.01 (m) which states “Each claim begins with a capital letter and ends with a period.”
Claim 12 recites “monocyte derived” which is a compound adjective and should be hyphenated as “monocyte-derived.”
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
Scope of Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-2, 4-5, 7-9, 11-12, and 14 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for:
A method of transfecting a human macrophage in vitro with genetic material, the method comprising the steps of:
contacting a human macrophage with genetic material;
electroporating the macrophage with a first pulse phase, wherein the first pulse phase comprises a burst of unipolar pulses or a square pulse, wherein each pulse is between 750-1000V, and wherein the first pulse phase lasts for a total period of between 20- 500μs; and
electroporating the macrophage with a second pulse phase, wherein the second pulse phase comprises a burst of pulses or a square pulse, wherein each pulse is between 50-225V, and wherein the second pulse phase lasts for a total period of 2000-50000μs,
does not reasonably provide enablement for methods of transfecting a human macrophage in vivo with genetic material.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to practice the method of the invention commensurate in scope with these claims.
Wands Factors
The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The Court in Wands states: “Enablement is not precluded by the necessity for some 'experimentation.'” Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. “Whether undue experimentation is needed is not a single simple factual determination, but rather is a conclusion reached by weighing many factual considerations.” (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all of these factors are considered, a sufficient amount for a prima facie case is discussed below.
Breadth of the Claims
Instant claims encompass:
Methods of “transfecting a human macrophage with genetic material”
This encompasses both in vitro and in vivo methods.
Amount of Direction or Guidance Presented
While instant claims encompass in vivo methods of transfecting comprising electroporation, no direction or specific guidance is presented for in vivo methods of transfecting comprising electroporation in the instant specification.
Absent Working Examples
While instant claims encompass in vivo methods of transfecting comprising electroporation, no working examples are provided for in vivo methods of transfecting comprising electroporation in the instant specification.
State of the Prior Art and Unpredictability of the Prior Art
in vivo Electroporation
Regarding in vivo electroporation, Applicant is first directed to the art of Somiari et al. (Mol Ther
. 2000 Sep;2(3):178-87.; henceforth “Somiari”). Somiari evidences critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage (abstract). Somiari evidences the flow of current through the extracellular electrolyte produces heat and associated cell damage which may result in reduced levels and/or a shorter period of gene expression (pg. 182 col. 2; Figure 2). Somiari evidences damage may occur by overheating cells (pg. 182 col. 2). Somiari evidences many optimized methods for transfection with free plasmid create significant tissue damage at the site of injection, and this may activate pathways or release factors required for direct transfection (pg. 183 col. 2). Somiari evidences future research must address and overcome the
mechanisms of toxicity, define the interaction between electroporative conditions and nucleic acid structure and size, and identify transcriptional elements that may be used to control the levels and distribution of transgene expression (pg. 185 col. 2 and pg. 186 col. 1).
Regarding in vivo electroporation, Applicant is additionally directed to the art of Luft et al. (J Biomol Screen. 2015 Sep;20(8):932–942.; henceforth “Luft”). Luft evidences transfection methods impose a stressful condition to the cells including induction of autophagy,
interference with translation mechanisms, and an induction of an interferon (IFN) response in mammalian cells and can result in toxicity and associated phenomenon (pg. 935). Luft evidences the duration and spacing of pulses, as well as the intensity, are crucial for high levels of uptake of macromolecules in cells and the main parameters that determine electropermeability are cellular factors such as cell density, cell architecture, and cell biochemistry, physicochemical factors such as temperature, pH, osmolarity, and ionic concentration of the buffer used and electrical parameters such as electrical field strength, voltage, pulse length, pulse number, and electrode geometry (pg. 935 col. 2). For in vivo electroporation specifically, Luft evidences when using electroporation in tissues such as solid
tumors, the electrical field and transmembrane voltage will be dependent on the tissue architecture and microenvironment of the cells, efficiency of electrotransfer in tissues is relatively low with only a few percentage of transfected cells, and uptake of large macromolecules such as DNA is generally limited to the outer layers, probably because of inefficient transport across longer distances (pg. 937 col. 2).
Therefore, because the arts of Somiari and Luft evidence specific parameters are required for tissue-specific in vivo electroporation, Somiari and Luft evidence transfection in vivo is unpredictable (low efficiency and limitation to the outer layers), Applicant is not enabled for in vivo methods of transfection of human macrophages comprising electroporation.
Unpredictability of the Prior Art and the Quantity of Experimentation Necessary
As the arts of Somiari and Luft demonstrate, the obstacles that hinder the use of the claimed method in in vivo embodiments are not easy tasks to be done or solely routine experimentation to enabled particular embodiments of the claimed method. The type of experimentation would require new methodologies. This level of experimentation goes beyond what would be routine optimization know at the time of filing. As such, the amount of experimentation would be undue.
The physiological art is recognized as unpredictable (MPEP 2164.03). As set forth in In re Fisher, 166 USPQ 18 (CCPA 1970), compliance with 35 USC 112(a) requires: “That scope of claims must bear a reasonable correlation to scope of enablement provided by specification to persons of ordinary skill in the art; in cases involving predictable factors, such as mechanical or electrical elements, a single embodiment provides broad enablement in the sense that, once imagined, other embodiments can be made without difficulty and their performance characteristics predicted by resort to known scientific laws; in cases involving unpredictable factors, such as most chemical reactions and physiological activity, scope of enablement varies inversely with degree of unpredictability of factors involved.” Moreover, the courts have also stated that reasonable correlation must exist between scope of exclusive right to patent application and scope of enablement set forth in the patent application (27 USPQ2d 1662 Ex parte Maize!.). In view of the foregoing, due to the lack of sufficient guidance provided by the specification regarding the issues set forth above, the state of the relevant art, and the breadth of the claims, it would have required undue experimentation for one skilled in the art to practice the instant broadly claimed invention.
Scope of Enablement - Conclusion
In conclusion, the breadth of the claims lack enablement because the specification provides no guidance or working examples for in vivo transfection methods comprising electroporation steps. The art at the time of effective filing fail to provide specific guidance that supplement to shortcomings of the specification and further teaches that the breadth of claims cannot predictably be done. Further, a great deal of new methodology would need to be developed to enable the full breadth of the claims and this level of experimentation is undue.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 8-9 and 11 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 8-9 and 11 recite “preferably” with no disclosed corresponding definition of “preferably.” If stated in the claims, examples and preferences may lead to confusion over the intended scope of a claim (see MPEP 2173.05(d). Therefore, it is unclear which aspect(s) of the claim is/are intended to be within the scope(s) of the claim(s) (see also MPEP 2173.05(b)).
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 7-8 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 7 recites “the burst of unipolar pulses are positive or negative pulses.” However, unipolar pulses may only be positive or negative, so claim 7 cannot further limit claim 1, upon which it depends.
Claim 8 recites “the genetic material comprises a nucleic acid, preferably DNA or RNA.” Since genetic material may only be nucleic acid, and DNA and RNA are the only types of nucleic acids that are genetic material, claim 8 cannot further limit claim 1, upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-2, 5, 8-9, 11-12, and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Martinet et al. (Biotechnol Lett. 2003 Jul;25(13):1025-9.; see IDS filed 25th, November, 2022; henceforth “Martinet”) in view of Zhang et al. (Methods Mol Biol. 2009:531:123-43.; henceforth “Zhang”), Demiryurek et al. (Biochim Biophys Acta. 2015 Aug;1848(8):1706-14. Epub 2015 Apr 22.; henceforth “Demiryurek”), and Sixto et al. (US 2018/0100161 A1; henceforth “Sixto”).
Regarding claim 1, Martinet discloses a method of transfecting human monocytes (human U937 or THP1 monocytes) with genetic material (purified pEGFPN3 DNA), the method comprising the steps of:
(a) contacting a human monocyte with genetic material (“100 µL of the cell suspension was mixed with 1µg purified pEGFP-N3 DNA);
electroporating the macrophage with a first pulse phase, wherein the first pulse phase is 750 V (within the claimed range of 750-1000V); and
(c) electroporating the human monocyte with a second pulse phase of 150 V (within the claimed range of (50- 225V) (pg. 1026 col. 1 3rd para. “Transfection”).
However, regarding claim 1, although Martinet teaches the teaches the technique is suitable not only for human THP-1 monocytes but also for other hard to transfect cells, Martinet is silent to using the method to transfect human macrophages.
Nevertheless, regarding claim 1, Zhang teaches methods of transfecting human monocytes or macrophages using electroporation (pg. 4-5; “human and mouse macrophage cell lines” Pg. 6 Note 2). Zhang teaches Macrophages have great potential as drug delivery vehicles because of their ability to home to inflamed tissue and delivery of exogenous gene products using genetically modified macrophages is an area with great therapeutic and/or immunomodulatory potential (Introduction; pg. 1). Zhang teaches electroporation parameters applicable to human THP-1 and U937 cell lines are also applicable to human and mouse macrophage cell lines (pg. 6 note 2).
Therefore, regarding claim 1, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method of Martinet, and simply substitute the known prior art element of the human macrophages of Zhang for the human THP-1 and U937 monocytes of Martinet to obtain the predictable result of a method for transfection human macrophages. One of ordinary skill would have been motivated to do so as taught by Zhang to prepare human macrophages as drug delivery vehicles for therapeutic or immunomodulatory purposes (Introduction; pg. 1). Regarding the reasonable expectation of success, Zhang evidences electroporation parameters applicable to human THP-1 and U937 cell lines are also applicable to human and mouse macrophage cell lines (pg. 6 note 2) and Zhang evidences relatively high efficiency gene expression in primary human or murine macrophages is routine (Summary; pg. 1).
However, regarding claim 1, although Martinet teaches two pulse phases (pg. 1026 col. 1 3rd para. “Transfection”), Martinet is silent to the waveform of the pulses.
Nevertheless, regarding claim 1, Zhang cites Martinet (pg. 6 section “3.2. Nucleofection”). Zhang teaches square wave pulses and exponential pulses as known alternatives for transfection of mammalian cells, and Zhang teaches electroporation devices that generate square wave pulses have been well established for the transfection of mammalian cells including human and mouse macrophage cell lines (pg. 6 Note 2).
Therefore, regarding claim 1, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method of Martinet, and combine the known prior art element of the square wave pulse of Zhang with the method of Martinet to obtain the predictable result of transfection of mammalian cells including human and mouse macrophage cell lines. Additionally, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method of Martinet and choose from the finite number of two predictable solutions of square wave pulses and exponential pulses presented by Zhang. Regarding the reasonable expectation of success, Zhang evidences successful electroporation of macrophages (pg. 6-7 “3.2. Nucleofection”) and Zhang further evidences uses of electroporation devices that generate square wave pulses are well established for the transfection of human macrophage cell lines (pg. 6 Note 2).
However, regarding claim 1, although Martinet teaches a two-pulse-electroporation protocol with a high-voltage first pulse followed by a low-voltage second pulse, Martinet is silent to a specific pulse length time for the transfection.
Additionally, regarding claim 1, Demiryurek teaches a two-pulse-electroporation protocol consisting of a short-duration 1 ms, high-voltage first pulse followed by a longer, 32 ms, low-voltage second pulse can increase delivery efficiency and preserve viability (abstract).
Therefore, regarding claim 1, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to practice the method as suggested by Martinet in view of Zhang, and combine the known prior art element of the short-duration timing of the first pule and longer duration of the second pulse of Demiryurek to obtain the predicable result of cell electroporation. One of ordinary skill would have been motivated to do so as taught by Demiryurek to increase delivery efficiency and preserve viability (abstract). Regarding the reasonable expectation of success, Demiryurek evidences successful transfection of cells with a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse (pg. 1707 “2.2. Electroporation protocol”).
Regarding the specific pulse lengths of the first and second pulses of claim 1, although Demiryurek teaches and makes obvious a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse, the prior art does not suggest the specific claimed pulse lengths of a total period of between 20-500μs for the first pulse phase and a total period of 2000-50000μs for the second pulse phase.
Nevertheless, regarding claim 1, Sixto teaches pore formation during electroporation can be controlled by adjusting various parameters, especially pulse length (para. [0004]).
Therefore, regarding claim 1, one of ordinary skill would have been motivated by routine procedure, as taught by Sixto, to adjust and optimize the pulse length in the suggested method to control pore formation during electroporation and one of ordinary skill would not consider the specifically claimed first and second pulse-lengths inventive.
Finally, regarding claim 1, Applicant is reminded that generally, differences in timings will not support patentability of subject matter encompassed by the prior art unless there is evidence indicating such timing is critical (MPEP 2144.05 II).
Regarding claim 2, further to the discussion of claim 1 above, as stated above (see claim 1 rejection above), Martinet teaches the first pulse phase is 750 volts, which is in the range of about 950 V (v).
Additionally, regarding claim 2, the taught value of first pulse phase is 750 volts is close to the value of 800 volts in the claimed range of between 800-1000V (i). Applicant is directed to MPEP section 2144.05 which states a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985) (Court held as proper a rejection of a claim directed to an alloy of "having 0.8% nickel, 0.3% molybdenum, up to 0.1% iron, balance titanium" as obvious over a reference disclosing alloys of 0.75% nickel, 0.25% molybdenum, balance titanium and 0.94% nickel, 0.31% molybdenum, balance titanium. "The proportions are so close that prima facie one skilled in the art would have expected them to have the same properties.").
Therefore, regarding claim 2, in the instant case, because the taught value of 750 V is so close to the claimed value of 800 V that prima facie one skilled in the art would have expected them to have the same properties as the instantly claimed value of 800 V, the claimed range of between 800-1000V (i) is prima facie obvious.
Regarding claim 5, further to the discussion of claim 1 above, as stated above (see claim 1 rejection above), Martinet teaches the second pulse phase is 150V (pg. 1026 col. 1 3rd para. “Transfection”), which is within the claimed ranges of between 80-200V (i), between 85-175V (ii), and between 90-150V (iii).
Regarding claims 8-9 and 11, further to the discussion of claim 1 above, as stated above (see claim 1 rejection above), Martinet teaches the genetic material comprises the nucleic acid DNA (pEGFP-N3 DNA; pg. 1026 col. 1 3rd para. “Transfection”) (instant claim 8) which comprises a nucleic acid encoding GFP, which is a heterologous gene of interest (GFP; pg. 1026 col. 1 3rd para. “Transfection”) (instant claim 9), and is comprised on a plasmid vector (pEGFP-N3 DNA; pg. 1026 col. 1 3rd para. “Transfection”) (instant claim 11).
Regarding claim 12, further to the discussion of claim 1 above, concerning the limitation that the macrophages are “human monocyte derived” this is product-by process language with respect to the macrophages. Applicant is reminded that product-by process claims are not limited by the manipulation of the recited steps, only the structure implied by the steps. “[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process.” In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) (see MPEP 2113). In the instant case, the human macrophages made obvious by Zhang above (see claim 1 rejection above), are structurally indistinguishable from human macrophages that are “human monocyte derived” and therefore the human macrophages suggested by Zhang meet instant claim limitations.
Regarding claim 14, further to the discussion of claim 1 above, Martinet discloses the cell density solution is 107 cells/mL which is within the claimed ranges of between 1x105 to 1x109 cells/mL (i), between 5x105 to 8x108 cells/mL (ii), between 1x106 to 6x108 cells/mL (iii), between 5x106 to 5x108 cells/mL (iv), and between 1x107 to 1x109 cells/mL (vi).
Hence, the claimed invention as a whole was prima facie obvious.
Examiner’s Remark
The subject matter of claims 4 and 7 are free of the prior art. However, claims 4 and 7 are not allowable because they is rejected under 35 U.S.C. 112a above, and are dependent on rejected claim 1. Claim 7 is also not allowable because it is rejected under 35 U.S.C. 112d above.
Conclusion
No claim is allowable.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRIANA N EBBINGHAUS whose telephone number is (703)756-4548. The examiner can normally be reached M-F 9:30 AM to 5:30 PM ET.
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/BRIANA N EBBINGHAUS/Examiner, Art Unit 1632 /VALARIE E BERTOGLIO/Primary Examiner, Art Unit 1632